During the current reporting period we focused on the following projects: 1) Studies of Pyrin and Yersinia pestis Previous studies from our laboratory demonstrated that inactivation of RhoA GTPase triggers a biochemical pathway leading to the assembly of the pyrin inflammasome, which activates the proinflammatory cytokines IL-1beta and IL-18. We have further shown that the Yersinia pestis YopE and YopT toxins inactivate RhoA and thereby stimulate pyrin inflammasome assembly, while the YopM Yersinia toxin blocks pyrin inflammasome activation by stimulating pyrin phosphorylation at residues 208 and 242 with subsequent binding of 14-3-3 inhibitory proteins to pyrin. In the present set of experiments, we explored the mechanism whereby YopM induces pyrin phosphorylation, and examined differences between wildtype pyrin and FMF-associated mutants in their susceptibility to YopM-mediated phosphorylation, both in knockin (KI) mice and in human peripheral blood leukocytes. We found that YopM not only induces pyrin phosphorylation by PKN1 and PKN2 (which we had shown previously), but also promotes the interaction of pyrin with RSK (ribosomal S6 kinase). Pyrin is not ordinarily a substrate for RSK, but in the presence of YopM RSK strongly phosphorylates pyrin at residues 208 and 242, thereby inducing 14-3-3 binding and blocking the pyrin inflammasome. However, if pyrin harbors an FMF-associated mutation, the interaction of YopM and pyrin is diminished. Furthermore, we found that (1) while Yersinia strongly suppresses IL-1beta production by wild type human and mouse leukocytes, this suppressive effect is much less substantial in mouse cells bearing FMF-associated mutations, and in human cells harboring biallelic or heterozygous FMF mutations; and (2) while Yersinia infection of wild type mice is lethal, mice harboring FMF mutations are protected. These data indicate that natural selection by the bubonic plague may well have contributed to the high FMF carrier frequencies observed in some populations. 2) DADA2 In the previous reporting period, we demonstrated the efficacy of tumor necrosis factor (TNF) inhibitors in preventing strokes in patients with DADA2. With continued follow up of the initial cohort of 15 DADA2 patients who had 55 strokes in a total of 2077 patient-months before anti-TNF initiation, there have been no strokes in 733 patient-months of observation since the initiation of TNF inhibitors. In collaboration with Peter Merkel and the Vasculitis Clinical Research Consortium, we have screened 117 patients with idiopathic PAN, all of whom tested negative for hepatitis B virus infection, for ADA2 mutations. Four (3.4%) had biallelic rare missense variants in ADA2 with a minor allele frequency of less than 0.005. Of the 7 distinct variants present in these 4 patients, 6 had previously been reported as causative for DADA2, and the remaining variant is computationally predicted to be damaging to protein function. Four additional patients were carriers for monoallelic variants, one of which has been reported in DADA2 before. Taken together, the treatment and screening data indicate that all new cases of non-hepatitis associated PAN should be tested for DADA2. 3) SIFD Our experience with 9 patients with biallelic mutations in TRNT1 was detailed in last year's report. A manuscript summarizing these findings was published online in the Annals of the Rheumatic Diseases in January 2018. 4) Evaluation of Putative New Autosomal Recessive Disorders Deficiency of USP43: We identified a patient from a founder Tatar population who presented with early-onset recurrent fevers, rash, subcutaneous nodules, lipodystrophy, and arthritis. Based on the clinical phenotype resembling chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), we conducted a candidate analysis of genes encoding proteasome components, which was negative. Whole-exome sequencing (WES) identified a novel predicted deleterious homozygous candidate variant within a poorly characterized deubiquitinase, USP43 (p.G837K), present only in the proband. The patient manifested a type I interferon (IFN) gene expression signature in peripheral blood, high basal levels of phospho-STAT1 in peripheral blood mononuclear cells (PBMCs), and increased serum levels of inflammatory cytokines. The patient has responded well to JAK inhibitors. We conducted knockdown experiments to evaluate the effect of USP43 on proteasome function. Depletion of USP43 led to increased levels of intracellular ubiquitinated proteins, which was rescued by the transfection of wild-type USP43. SHARPENIA: The linear ubiquitin chain assembly complex (LUBAC) comprises HOIL-1, HOIP, and SHARPIN. Patients with HOIL-1 and HOIP deficiencies have been reported to have immunodeficiency, autoinflammation, and amylopectinosis. Although mice deficient in Sharpin have severe dermatitis, the role of SHARPIN in human disease is unknown. We investigated a 14 year-old boy from a consanguineous family in India with early-onset purulent polyarthritis, parotitis, hepatomegaly, and colitis, but no history of dermatitis or severe infection. By WES we identified a homozygous frameshift mutation in SHARPIN in the proband (c.220dupC). Other family members were carriers for this variant and unaffected. The patient's PBMCs had no detectable SHARPIN protein. Mutant cells displayed impaired canonical NF-kappaB activity. In contrast to HOIL-1 and HOIP deficiencies, the patient's monocytes did not show hyper-responsiveness to IL-1beta. Based on the TNF-dependence of inflammation in Sharpin-deficient mice, we initiated etanercept, with dramatic salutary effect. To investigate the role of SHARPIN in bone, we knocked out SHARPIN in a human immortalized osteoblast line. Despite attenuated NF-kappaB activity in PBMCs, the SHARPIN knockout osteoblasts secreted higher levels of IL-6, IP10/CXCL10, and RANTES/CCL5 after IL-1beta stimulation than control cells. Deficiency of OTUD5: We have identified 6 male patients with novel hemizygous missense mutations in an X-linked gene OTUD5, which encodes a Lys48/Lys63-chain-specific deubiquitination enzyme previously not linked to human disease. Conditional knockout in murine CD4 T lymphocytes is known to cause an inflammatory phenotype. We observed structural brain malformations, congenital heart disease, ambiguous genitalia, post-axial polydactyly, arthrogryposis, craniofacial defects, and/or hematopoietic abnormalities among affected patients. Disease-causing variants result in decreased OTUD5 function by reducing overall protein expression and by altering enzymatic activity. Consistent with a loss of function phenotype, decreasing OTUD5 levels causes aberrant cellular differentiation in vitro and in vivo and leads to altered RNA expression in patient-derived cells. The OTUD5 substrates UBR4 and UBR5 are destabilized in OTUD5 depletion/deletion models. 5) Patients with Mutations in Previously Reported Disease Genes RNF31 encodes HOIP, the catalytic subunit of LUBAC. The only reported patient with HOIP deficiency manifested immunodeficiency, autoinflammation, and amylopectinosis. In a child with autoinflammation and common variable immunodeficiency, we found biallelic loss-of-function mutations in RNF31. Serum IL-6 was significantly increased, and there was a type I IFN gene expression signature in PBMCs. NCF1 encodes the p47phox component of the NADPH oxidase complex. While loss-of-function mutations cause chronic granulomatous disease, carriers of the p.R90H missense variant have been shown to be at increased risk for adult-onset systemic lupus erythematosus. By WES, we identified a child with a recurrent fever syndrome and type I IFN signature who is homozygous for this variant, with antinuclear antibodies but lacking other clinical manifestations of lupus.